Biotechnology-developing scalable production processes for recombinant adeno-associated virus.[unreadable] To extend our efforts of therapeutic gene transfer beyond in vitro studies and small animal models, the impediment of using adherent cells for rAAV production had to be overcome. The production of biologically active, recombinant dependovirus in invertebrate cells originated in this laboratory following a critical and unexpected experimental result demonstrating that AAV Rep 78 supported AAV DNA replication in Sf9 cells. Although it was known that mammalian virus structural proteins assemble into virus-like particles (VLPs) in insect cells, e.g. Papillomaviridae, virus or vector DNA replication and packaging was never examined in invertebrate cells. Producing other recombinant virus vectors devoid of virus genes appears possible especially for non-enveloped, DNA viruses.[unreadable] Similar to rAAV production in mammalian cells, insect cells require both cis and trans factors from AAV for replication, packaging, and particle formation. The AAV terminal palindromes, referred to as the inverted terminal repeats (ITRs), function as primers for DNA synthesis and are considered important for encapsidation. Replication and packaging require two of the four AAV non-structural proteins: one of the p5 proteins, either Rep 78 or Rep 68, and one of the p19 proteins, either Rep 52 or Rep 40.[unreadable] Currently, we are able to produce AAV serotypes 1, 2, 4, 5, 6, and 8 vectors using BEVs in insect cells. Newly developed improvements to production processes utilizing on-line permittivity measurements, multiplicity of infection analysis, cell density at time of infection, and baculovirus stability studies, have increased both our understanding of the production process as well as the yields of rAAV. We now obtain &#8805; 3x1014 particles /l of rAAV, thus a 350 l production run may yield &#8776; 1017 rAAV particles, sufficient for toxicological studies, and preclinical, dose escalation experiments in large animal models.[unreadable] Importantly, we have validated therapeutic rAAV vectors produced with the insect cell system in small animal models of neurodegenerative diseases (such as spinocerebellar ataxia and Huntingtons disease) and cancer. These advances in large-scale AAV production will allow us to address specific catastrophic, intractable human diseases (such as Duchenne muscular dystrophy) for which large amounts of recombinant vector are essential for successful outcome.